Comprehensive Synthesis of Substrates, Intermediates, and Products of the Sulfoglycolytic Embden-Meyerhoff-Parnas Pathway

Article abstract of DOI:10.1021/acs.joc.9b00055

Sulfoglycolysis is a metabolic pathway dedicated to the catabolism of the sulfosugar sulfoquinovose (SQ) into smaller organosulfur fragments. An estimated 10 billion tonnes of SQ fluxes through sulfoglycolysis pathways each year, making it a significant aspect of the biogeochemical sulfur cycle. Delineating the molecular details of sulfoglycolysis requires authentic samples of the various metabolites in these pathways. To this end, we have established chemical and chemoenzymatic methods for the synthesis of the key organosulfur metabolites sulfoquinovosylglycerol, SQ (also in ¹³C₆-labeled form), sulfofructose, sulfofructose-1-phosphate, sulfolactaldehyde, and 2,3-dihydroxypropanesulfonate, as well as an improved route to the chromogenic sulfoquinovosidase substrate 4-nitrophenyl α-sulfoquinovoside.

Full text of DOI:10.1021/acs.joc.9b00055

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Article
Comprehensive synthesis of substrates, intermediates and
products of the sulfoglycolytic Embden-Meyerhoff-Parnas pathway
Palika Abayakoon, Ruwan Epa, Marija Petricevic, Christopher Bengt, Janice
W.-Y. Mui, Phillip L. van der Peet, Yunyang Zhang, James P. Lingford,
Jonathan M White, Ethan D. Goddard-Borger, and Spencer J. Williams
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Feb 2019
Downloaded from http://pubs.acs.org on February 11, 2019
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Comprehensive synthesis of substrates, intermediates and products of the
sulfoglycolytic Embden-Meyerhoff-Parnas pathway
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Palika Abayakoon,† Ruwan Epa,† Marija Petricevic,† Christopher Bengt,† Janice W.-Y.
Mui,† Phillip L. van der Peet,† Yunyang Zhang,† James P. Lingford,‡ Jonathan M. White,†
Ethan D. Goddard-Borger,‡§ Spencer J. Williams*†
†School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University
of Melbourne, Parkville, Vic 3010
‡
ACRF Chemical Biology Division, The Walter and Eliza Hall Institute of Medical
Research, Parkville, Victoria 3010, Australia
§Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010,
Australia
Email: sjwill@unimelb.edu.au
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Abstract
Sulfoglycolysis is a metabolic pathway dedicated to the catabolism of the sulfosugar
sulfoquinovose (SQ) into smaller organosulfur fragments. An estimated 10 billion tonnes of
SQ fluxes through sulfoglycolysis pathways each year, making it a significant aspect of the
biogeochemical sulfur cycle. Delineating the molecular details of sulfoglycolysis requires
authentic samples of the various metabolites in these pathways. To this end, we have
established chemical and chemoenzymatic methods for the synthesis of the key organosulfur
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metabolites sulfoquinovosylglycerol (SQGro), SQ (also in C -labelled form), sulfofructose
6
(SF),
sulfofructose-1-phosphate
(SFP),
sulfolactaldehyde
(SLA)
and
2,3-
dihydroxypropanesulfonate (DHPS), as well as an improved route to the chromogenic
sulfoquinovosidase substrate 4-nitrophenyl -sulfoquinovoside.
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INTRODUCTION
The sulfoglycolipid sulfoquinovosyl diacylglyceride (SQDG) is a significant chemical species
in the global biogeochemical sulfur cycle,1-2 along with the sulfur-containing amino acids
cysteine and methionine, and the osmolytes dimethylsulfoniopropionate3 and
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dimethylsulfoxonium propionate. The abundance and ubiquity of SQDG has fostered the
evolution of specialised microbial pathways for the catabolism of its unusual sulfoquinovose
(SQ, 1) head-group. Benson first proposed the existence of sulfoglycolytic pathways in 1961;
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named by analogy with the glycolytic processes for glucose metabolism. Two sulfoglycolytic
processes capable of catabolizing SQ have since been described.6-7 In both cases,
sulfoglycolysis is preceded by liberation of SQ from its glycosides SQDG and its delipidated
8-9
forms lyso-SQDG and -sulfoquinovosyl glycerol (SQGro, 2), and rapid equilibration of SQ
anomers by an SQ mutarotase to overcome the intrinsically slow uncatalyzed mutarotation of
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6, 11
this sugar. The first sulfoglycolysis pathway was identified in Escherichia coli.
broad similarity with the classical Embden-Meyerhoff-Parnas (EMP) glycolysis pathway
which converts glucose to ATP and pyruvate) and thus is referred to as the sulfoglycolytic
It shares
(
EMP (sulfo-EMP) pathway. The sulfo-EMP pathway consists of a series of enzymes that
catalyse isomerization of SQ to sulfofructose (SF, 3; YihS, SQ-SF-isomerase), phosphorylation
of SF to provide sulfofructose-1-phosphate (SFP, 4; YihV, SF-kinase), retro-aldol cleavage of
SFP to dihydroxyacetonephosphate (DHAP; which enters central carbon metabolism) and
sulfolactaldehyde (SLA, 5; YihT, SFP-aldolase), and finally reduction of SLA to S-2,3-
6
dihydroxypropanesulfonate (S-DHPS, 6; YihU, SLA-reductase) (Figure 1). This system is
under control of a transcriptional regulator (CsqR), which represses expression of
sulfoglycolytic proteins in the absence of SQ.12 An alternative sulfoglycolytic pathway
mimicking the Entner-Douderoff pathway has also been identified, as first described in
Pseudomonas putida SQ1.7
Biochemical and structural studies of the sulfo-EMP pathway would benefit from the
development of methods to synthesize and purify the various substrates, intermediates and
products of the pathway. Current methods for the synthesis of SQGro are iterations of an
1
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arduous procedure first developed by Miyano and Benson and later modified by Roy and
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13-16
Hewlins. Several syntheses of SQ have been reported,
and while these methods are
sufficient for the preparation of small amounts of material, larger quantities are needed to
support metabolic studies. A two-step method reported for the synthesis of the chromogenic
SQase substrate, 4-nitrophenyl -sulfoquinovoside (PNPSQ, 7), while direct, is challenging in
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practice due to the poor stability of a thioacetate intermediate. To our knowledge no methods
for the synthesis of pure samples of SF, SFP or SLA have been reported (for previous efforts
see: SFP, SLA5, 7). Finally, while there are several reports for the synthesis of racemic
DHPS,17-18 these are arduous and time-consuming, and do not provide effective ways to purify
the final product. As part of ongoing efforts to understand the molecular details of the sulfo-
EMP pathway, we herein disclose improved methods for the synthesis of the known
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compounds SQGro, PNPSQ, SQ (also in C-labelled form) and DHPS, and the first syntheses
of the sulfoglycolytic intermediates SF, SFP and SLA.
SQGro (2)
SO3
SQ (1)
SO3
SF (3)
SFP (4)
SLA (5)
DHPS (6)
YihQ
YihR
OH
SO3

2
OPO3
OH
SO3
O
O
OH
O
OH
OH
YihS
YihV
YihT
OH
YihU
OH
O
O
YihO
O
SO3
SO3
YihP
HO
HO
OH
HO
OH
OH
OH
ATP ADPHO
OH
NADH NAD+
HO
HO
HO
OH
OH
O
OPO32

HO
OPO32
pyruvate
ATP
HO
OH
HO
NADH NAD+
Gro
ATP ADP
DHAP
E. coli
Figure 1. The sulfoglycolytic Embden-Meyerhof-Parnas pathway for metabolism of
sulfoquinovosylglycerol (SQGro) and sulfoquinovose (SQ) in E. coli. YihO and YihP are putative
influx/efflux proteins.
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Results and discussion
Synthesis of the SQ glycosides SQGro and PNPSQ. Miyano and Benson reported the
synthesis of SQGro in a multi-step process involving the preparation of SQ from 1,2-O-
isopropylidene--D-glucofuranose in three steps, conversion to the allyl -sulfoquinovoside,
permanganate oxidation and multiple ion-exchange steps that ultimately provided the
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cyclohexylammonium salt. Roy and Hewlins applied the same general approach, but made
some improvements to the final purification through the isolation of the brucine salt prior to
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19-
conversion to the cyclohexylammonium salt. Inspired by approaches to related molecules,
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we instead sought to utilize commercially available allyl -D-glucopyranoside 8 as a
precursor, as it possesses the underlying carbon framework of the target. Accordingly, Appel
bromination of the 6-position of 8 afforded the bromide 9 in good yield (Scheme 1A). Direct
substitution of the bromide with sodium sulfite in water afforded the known allyl -D-
sulfoquinovoside 10.13-14 While prior syntheses employ KMnO to dihydroxylate this alkene,
13-
4
1
4
19
we were inspired by related work using catalytic OsO and Me NO to adopt a ligand-
4
3
catalyzed Sharpless dihydroxylation, which provides a rate enhancement for the oxidation of
21
terminal alkenes versus osmium tetroxide alone. We were, however, aware of the limited
ability of chiral catalysts used in related transformations of unprotected allyl -glycosides to
impart any meaningful diastereoselectivity on the dihydroxylation reaction.22 While
preliminary experiments with commercially available AD-mix reagents provided a successful
transformation, we had difficulty purifying the highly polar SQGro products from the large
amounts of salts derived from the potassium ferricyanide co-oxidant. In the spirit of the original
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1
Sharpless asymmetric dihydroxylation process, we therefore applied an Upjohn-type process
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3
using N-methylmorpholine N-oxide (NMO) as terminal oxidant but with K OsO . Treatment
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4
of 10 with catalytic K OsO , (DHQD) PHAL ligand, and NMO smoothly provided SQGro,
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and facilitated simple purification to provide the sodium salt in a 1:1 ratio of the 2'R and 2'S-
isomers. Benson has reported that the cyclohexylammonium salt can be recrystallized to
13
provide the pure 2'R-stereoisomer. Therefore we converted the sodium salt to the sulfonic
+
acid by passage through a column of Dowex-50 H resin, added cyclohexylamine to neutralize
the eluent, and concentrated the crude salt. Repeated fractional crystallization afforded a pure
diastereoisomer, with melting-point and NMR data matching that reported previously.13
Benson identified this material as the 2'R-stereoisomer by mixed melting point analysis with
the cyclohexylammonium salt of naturally derived material, for which the stereochemistry was
defined by use of the radiotracer method with co-precipitation with authentic L-, but not D-
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glycerate, a conclusion subsequently confirmed by X-ray analysis of the rubidium salt of
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naturally-derived SQGro. We confirmed the assignment of our synthetic material as 2'R-
SQGro through direct single crystal X-ray crystallographic analysis of the recrystallized
material (Scheme 1A, Inset; Figure S1).
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A
SO Na
O
SO Na
O
3
X
3
Na SO3
K OsO , DHQD PHAL
2 4 2
2
O
HO
HO
HO
HO
HO
HO
OH
2'
HO
H O, 100 °C
2
HO
O
NMO, t-BuOH/H O
2
HO
O
SQGro (2)
OH
O
67%
91%
CBr , PPh
pyr, 58%
8 X = OH
9
X = Br
10
4
3
2'R/2'S = 1:1
1
. Dowex-50 H+
. C H11NH2
SO H.C H11NH2
3
6
2
6
O
HO
HO
OH
HO
3
. crystallization from EtOH
O
OH
14%
R
2'R-SQGro (2)
Single crystal X-ray of
'R-SQGro.C H11NH2
2
6
B
OH
O
SBz
SO K
3
BzSH, DIAD
O
Oxone
O
HO
HO
HO
HO
HO
HO
HO
HO
KOAc, AcOH
1%
HO
Ph P, THF
OPNP
3
OPNP
OPNP
6
81%
1
1
12
PNPSQ (7)
Scheme 1. Synthesis of sulfoquinovosylglycerol (SQGro) and 4-nitrophenyl -D-
sulfoquinovoside (PNPSQ). Inset: thermal ellipsoid plot for 2'R-SQGro, ellipsoids are at
the 50% probability level.
We have previously reported the synthesis of PNPSQ (7) from PNPGlc (11), through a
two-step procedure involving Mitsunobu reaction with thioacetic acid, followed by oxidation
8
with KOAc/AcOH buffered mCPBA. However, this procedure suffered from poor stability of
the intermediate thioacetate, leading to variable yields for the subsequent oxidation. Careful
analysis of reaction mixtures suggested that part of the reason for the instability of the
thioacetate was its facile hydrolysis to the thiol and oxidation to the disulfide. Additionally,
during the oxidation reaction, the acetyl group was found to migrate to the 4-position, to yield
small amounts of 4-O-acetyl-PNPSQ. We sought to improve this reaction by use of thiobenzoic
acid, which we anticipated may provide a more stable thioester that should be less prone to
acyl migration in the oxidation step. Accordingly, Mitsunobu reaction of 11 with thiobenzoic
acid afforded the thiobenzoate 12 in 81% yield (Scheme 1B). While this could be oxidized to
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PNPSQ with KOAc/AcOH buffered mCPBA, better yields were obtained with Oxone in
2
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AcOH, affording PNPSQ in 61% yield.
Synthesis of SQ. Several syntheses of SQ have been reported, which use either 1,2-O-
isopropylidene--D-glucofuranose13-15 or methyl glucopyranoside as starting materials, with
the sulfonate group introduced directly by nucleophilic substitution of a leaving group with
sulfite. While direct, these methods provide products that can be difficult to purify. We herein
report a longer route, but one that in our hands has proven to be easily scaled to multigram
quantities of SQ, and through which the use of protecting groups enables purification of
intermediates by crystallization or silica gel chromatography. Using the method of Garegg and
16
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Sameulsson (I , Ph P, imidazole; then Ac O, pyr), methyl -D-glucopyranoside was converted
2
3
2
to the iodide 13,27 then substituted with potassium thioacetate to afford the thioester 14
(Scheme 2A). Oxidation with Oxone afforded the protected sulfonate 15, and treatment with
NaOMe/MeOH allowed deprotection to give the methyl glycoside 16. Finally, SQ (1) was
obtained by treatment with aqueous HCl.
A
OH
O
I
SAc
O
1
. I , Ph P, imid
2 3
KSAc
O
HO
HO
AcO
AcO
AcO
AcO
HO
2. Ac O, py
AcO
3
acetone
quant.
AcO
14
2
OMe
AcO
OMe
OMe
1
[ref. 27]
SO K
SO K
3
3
SO K
3
HCl
H O
Oxone
94%
NaOMe
O
O
HO
O
HO
HO
AcO
HO
MeOH
98%
AcO
HO
2
OH
HO
OMe
OMe
quant.
15
16
SQ (1)
= 13C
. I , Ph P, imid
B
OH
O
I
SAc
1
KSAc
2
3
O
O
HO
HO
AcO
AcO
AcO
AcO
HO
AcO
18
10:1, 
OMe
acetone
3%
AcO
19
20:1, 
OX
2. Ac O, py
2
OMe
8
34%, two steps
AcCl
X = H
MeOH
17 X = Me
SO K
SO K
3
SO K
3
HCl
Oxone
3
K2CO3
O
O
HO
21
HO
O
HO
HO
AcO
HO
AcOH/KOAc
84%
AcO
MeOH
9%
H O
2
HO
OH
OMe
AcO
OMe
8
73%
13
2
0
( C )-SQ (22)
6
2
5:1, 
30:1, 
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Scheme 2. Synthesis of sulfoquinovose (SQ) and ( C )-SQ.
6
1
3
A modified version of this approach was used for the synthesis of ( C )-SQ (22).
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( C )-Glucose was converted to a mixture of methyl glucopyranosides 17 by treatment with
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HCl in MeOH at reflux. Next, iodination using I , Ph P and imidazole in toluene, followed
2
3
by work-up and then acetylation and recrystallization, afforded the iodide 18. Substitution with
KSAc (19), oxidation with Oxone (20), deacetylation with K CO (21) and finally acid-
2
3
1
3
catalyzed glycoside hydrolysis afforded ( C )-SQ (22).
6
Synthesis of SF and SFP. For the synthesis of SF, we were attracted to a key intermediate, the
bisacetal 23, which can be prepared in one step from D-fructose, as reported by Hong and co-
2
8
workers. Treatment of D-fructose with MeOH/TsOH, and then 2,2-dimethoxypropane (2,2-
DMP), afforded 23 in 64% yield (Scheme 3A). Compound 23 was treated with thiobenzoic
acid under Mitsunobu conditions to afford the thiobenzoate 24, and this was oxidized with
AcOH/KOAc buffered Oxone, to afford the sulfonate 25. Finally, careful hydrolysis of the
protected sulfonate in AcOH/water afforded SF (3) in quantitative yield. SF exists as a 3.7:1
1

: ratio of anomers, as determined by H NMR spectroscopy.
A
OMe
O
OMe
O
O
O
O
BzSH, DIAD
Ph P, THF
HO
HO
2,2-DMP, MeOH
TsOH
HO
BzS
OH
O
O
HO
OH
HO
3
HO
64%
9
1%
D-fructose
23
24
OMe
O
OH
O
AcOH, H O
O
Oxone
2
KO S
KO S
3
3
O
OH
AcOH/KOAc
0%
quant.
HO
HO
OH
9
25
SF (3)
B
sulfofructokinase
ATP, MgCl2
OH
OH
OH
O
O
KO S
NEt .HO S
3 3
3
OH
OPO H Et N
3 2. 3
Tris pH 7,5
HO
HO
OH
54%
3
SFP (4)
Scheme 3. Synthesis of sulfofructose (SF) and sulfofructose-1-phosphate (SFP).
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For SFP (4) we utilized an enzymatic approach catalysed by E. coli SF kinase YihV.
YihV was expressed from a pET plasmid as a His -fusion and purified by immobilized metal
6
ion affinity chromatography. Incubation of a solution of SF (3) with 2 equivalents of ATP and
MgCl in the presence of YihV afforded a solution containing SFP (Scheme 3B). This was
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
purified using ion-paired HLPC with triethylammonium bicarbonate buffer, affording 4 as the
1
bis(triethylammonium) salt, as determined by H NMR spectroscopy. SF exists as a 4:1 :
ratio of anomers.
Synthesis of SLA. For the synthesis of SLA (5) we were inspired by processes used for the
preparation of glyceraldehyde-3-phosphate. These prepare the diethyl acetal for long term
storage, and then liberate the glyceraldehyde on demand through a brief treatment with acid
29
resin. Accordingly, we treated glycidol diethyl acetal (26) with sodium sulfite in water at
reflux, which provided the diethyl acetal 27 after silica gel chromatography and ion-exchange
(
Scheme 4). To convert 27 to SLA, a solution of the diethyl acetal in H O or D O was treated
2 2
+
at 80 °C with Dowex 50 H resin. The resulting SLA was determined to be a hydrate, as
indicated by a characteristic singlet at  4.44 ppm. No H/D exchange was noted at C2 over the
period of several days.
1
. Na SO , H O, 100 °C
OH
2
3
2
O
2. silica gel, Et N
O
SO Na
3
3
O
O
3. Dowex 50 Na+
O
26
27
quantitative
OH
Dowex 50 H+
HO
SO H
3
H O, 80 °C
OH
SLA-hydrate (5)
2
quantitative
Scheme 4. Synthesis of sulfolactaldehyde (SLA).
Synthesis of DHPS. Friese prepared DHPS in a direct, one-step fashion by treatment of allyl
17
18
alcohol with Ac O and H SO , and this procedure has been modernized by Mayer et al.
2
2
4
However, these procedures have a complex work-up involving multiple steps with evaporation
of Ac O, water, conversion to the barium salt and back to the free acid before careful titration
2
to the sodium salt, with no purification steps. While we were able to successfully reproduce
this procedure, the lack of purification at the end left doubt as to the purity of the isolated
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
material. Consequently, we sought to simplify this approach and to develop a crystallization
process. We were attracted to a short remark in an early paper by Benson who noted the
3
0
potential for formation of a cyclohexylammonium salt. After some experimentation we were
able to reproduce this observation and confirm that this salt is crystalline, and here provide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
complete details. Our preferred process involves heating a solution of allyl alcohol, Ac O and
2
1
8
H SO as described by Mayer et al., then evaporation of the solvent and co-evaporation with
2
4
H O until the pH of the distillate reached pH 6 (Scheme 5A). The residue is dissolved in EtOH
2
and neutralized with cyclohexylamine and can be directly crystallized from EtOH/toluene to
afford the pure cyclohexylammonium 2,3-dihydroxypropanesulfonate. While the yield is
modest at 40%, the low cost of the reagents and the ease of the process readily allows
preparation of decagram quantities.
A
OH
1
. Ac O, H SO
2 2 4
HO
HO
SO H.C H11NH2
3 6
2
. cyclohexylamine
DHPS (6)
40%
B
1. Na SO , H O, 100 °C
2 3 2
2. Dowex-50 (H+)
OH
O
HO
HO
SO H.C H11NH2
3
6
3
. cyclohexylamine
DHPS (6)
58%
Scheme 5. Synthesis of 2,3-dihydroxypropanesulfonate (DHPS).
We also devised an alternative approach for the synthesis of DHPS, in which glycidol
1
2
is treated with sodium sulfite in water at reflux (Scheme 5B). Passage of the reaction mixture
+
through a Dowex-50 H column is used to remove excess sulfite as sulfurous acid, and then
conversion to the cyclohexylammonium salt allows isolation of the product in 58% yield.
Conclusions
The sulfoglycolytic Embden-Meyerhof-Parnas pathway is a major conduit through which
organosulfur cycles in the terrestrial biosphere. However, detailed molecular studies have not
been possible owing to the lack of methods to synthesize the substrates, intermediates and
products of this pathway. The present disclosure of improved and in some cases new methods
1
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for the preparation of this complete series of compounds will assist future studies to understand
how SQ is processed by enzymes in this pathway.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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7
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9
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9
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Experimental
General
Pyridine was distilled over KOH before use. Dichloromethane and THF were dried over alumina
according to the method of Pangborn et al.31 Reactions were monitored using thin-layer
chromatography (TLC), performed with silica gel 60 F254. Detection was effected by charring in a
mixture of 5% sulfuric acid in methanol, 10% phosphomolybdic acid in EtOH, and/or visualizing with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
UV light. Flash chromatography was performed according to the method of Still et al. using silica gel
−
1
2
−1
6
0. Melting points were determined by a capillary apparatus. [α] values are given in deg 10 cm g .
D
NMR experiments were conducted on 400 and 500 MHz instruments, with chemical shifts referenced
1
1
relative to residual protiated solvent and are in parts per million (ppm). H− H COSY and HMQC
spectra were used to confirm proton and carbon assignments, respectively. Mass spectra were acquired
in the ESI-QTOF mode.
1
. Synthesis of SQGro
Allyl 6-bromo-6-deoxy-α-D-glucopyranoside (9). Carbon tetrabromide (2.10 g, 6.40 mmol) and
triphenylphosphine (1.90 g, 7.20 mmol) were added to a solution of allyl α-D-glucopyranoside 8 (1.00
o
g, 4.50 mmol) in dry pyridine (12 mL) at 0 C. The solution was warmed to room temperature and was
stirred overnight under a nitrogen atmosphere. After the completion of the reaction, methanol (2 ml)
was added and the solvent was removed under reduced pressure to give a gum. The gum was partitioned
between toluene (25 mL) and water (25 mL). The aqueous phase was collected, and the toluene phase
was extracted with water (10 mL). The combined aqueous layers were washed with additional toluene
(10 mL). The combined aqueous layers were then evaporated to give a gum that was purified by flash
chromatography (75-100% EtOAc/hexanes). Evaporation of the eluent yielded 9 as an off-white solid
0.710 g, 58%). m.p. 80-82 °C; []D25 +114.9 ° (c 0.47, CH OH); H NMR (400 MHz, CD OD) δ 6.04–
1
(
3
3
5
.90 (1 H, m, H2'), 5.34 (1 H, d, J2',3'trans = 18.2 Hz, H3'a), 5.18 (1 H, d, J2',3'cis = 10.5 Hz, H3'b), 4.83 (1
= 13.0, J1'a,2' = 5.1 Hz, H1'a), 4.05 (1 H, dd, J1'b,2 = 6.1 Hz,
H, d, J = 3.8 Hz, H1), 4.26 (1 H, dd, J
1'a,1'b
1
,2
H1'b), 3.78–3.68 (2 H, m, H5,6a), 3.65 (1 H, t, J2,3 = J3,4 = 9.3 Hz, H3), 3.52 (1 H, dd, J6a,6b = 10.4, J5,6a
13
1
=
6.4 Hz, H6b), 3.40 (1 H, dd, H2), 3.24 (1 H, t, J4,5 = 9.1 Hz, H4); C{ H} NMR (101 MHz, CD OD)
3
δ 134.0 (C2'), 116.4 (C3'), 97.7 (C1), 73.4 (C3), 72.5 (C4), 72.0 (C2), 71.3 (C5), 67.9 (C1'), 32.9 (C6);
–
HR-ESI-MS calcd for C H BrO [M+HCO ] 327.0085, found 327.0057.
1
0
16
5
2
Sodium allyl 6-deoxy-6-sulfonato-α-D-glucopyranoside (10). A solution of anhydrous sodium sulfite
0.937 g, 7.40 mmol) and compound 9 (0.500 g, 1.86 mmol) in water (15 mL) was heated under nitrogen
(
at reflux for 5 h. The solution was cooled to room temperature and the solvent was evaporated under
reduced pressure. A slurry of the resulting white solid in methanol (10 mL) was stirred overnight,
filtered and the filtrate evaporated to give a crude residue (0.700 g). The residue was purified by flash
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chromatography (27:2:1 to 6:2:1 EtOAc/MeOH/H O), to give 10 as a clear viscous oil (0.380 g, 67%
2
yield). []D25 +94.9° (c 0.32, CH OH); H NMR (400 MHz, CD OD) δ 6.04–5.92 (1 H, m, H2'), 5.38
1
3
3
(
1 H, d, J2',3'trans = 18.7 Hz, H3'a), 5.15 (1 H, d, J2',3'cis = 11.5 Hz, H3'b), 4.81 (1 H, d, J = 3.8 Hz, H1),
1,2
4
9
.40 (1 H, dd, J1'a,1'b = 12.9, J1'a,2 = 5.2 Hz, H1'a), 4.14–4.01 (2 H, m, H5,1'b), 3.67 (1 H, t, J = J3,4 =
2,3
.3 Hz, H3), 3.42 (1 H, dd, J =3.8 Hz, H2), 3.35 (1 H, dd, J
= 14.4, J5,6a = 2.1 Hz, H6a), 3.10 (1
1
,2
6a,6b
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
H, t, J4,5 = 9.4 Hz, H4), 2.94 (1 H, dd, J5,6b = 8.9 Hz, H6a); C{ H} NMR (101 MHz, CD OD) δ 134.2
3
(C2'), 116.4 (C3'), 97.1 (C1), 73.7 (C4), 73.5 (C2), 72.0 (C3), 68.2 (C5), 67.7 (C1'), 53.0 (C6); HR-ESI-
–
MS calcd for C H O S [M ] 283.0488, found 283.0481.
9
15
8
Sodium 2'R/S-glyceryl 6-deoxy-6-sulfonato-α-D-glucopyranoside (2). A solution of 10 (0.300 g,
.980 mmol) in water (5 mL) was added to a stirred mixture of K OsO .2H O (0.007 g, 0.0196 mmol),
0
2
4
2
(DHQD) PHAL (0.031 g, 0.0392 mmol), N-methylmorpholine N-oxide (0.230 g, 1.96 mmol) in t-
2
butanol/water (4:5) (9 mL). The resulting mixture was stirred under nitrogen for 24 h. The reaction was
quenched by the addition of anhydrous sodium sulfite (0.246 g, 1.96 mmol), and the mixture was stirred
for 30 min. The solvent was evaporated under reduced pressure and the crude residue was subjected to
flash chromatography (EtOAc/MeOH/H O, 8:2:1 to 5:2:1), to give 2 as a gum, 1:1 mixture of
2
1
diastereoisomers (0.300 g, 91%). H NMR (600 MHz, CD OD) δ 4.77 (1 H, d, J = 4.1 Hz, (H1-2'R),
3
4
.76 (1 H, d, J = 4.1 Hz, (H1-2'S), 4.11–3.99 (4 H, m, H5,1'a-2'R,5,1'a-2'S), 3.92–3.85 (1 H, m, H2'-
2'R), 3.83–3.78 (1 H, m, H2'-2'S), 3.71 (1 H, dd, J = 11.4, 5.4 Hz, H3'b-2'S), 3.66–3.48 (6 H, m,
H3,3'a,3'b-2'R,3,3’a,1'b-2'S), 3.41 (2 H, ddd, J = 9.7, 3.8, 1.7 Hz, H2-2'R,2-2'S), 3.36–3.32 (3 H, m,
H6a-2'R,6a-2'S,1'b-2'R), 3.10–3.03 (2 H, m, H4-2'R,4-2'S), 2.93–2.87 (2 H, m, H6b-2'R,6b-2'S);
1
3
1
C{ H} NMR (101 MHz, CD OD) δ 98.7, 98.2 (2 C, C1-2'R/S), 73.7, 73.6 (2 C, C3-2'R/S), 73.4, 73.3
3
(2 C, C4-2'R/S), 72.1 (2 C, C2-2'R/S), 71.0, 70.8 (2 C, C2'-2'R/S), 69.3 (C1'-2'R), 68.5, 68.4, 68.3 (3 C,
C1'-2'S,5-2'R/S), 62.8, 62.7 (2 C, C3'-2'R/S), 52.7, 52.6 (2 C, C6-2'R/S); HR-ESI-MS calcd for
–
C H O S [M ] 317.0542, found 317.0536.
9
17 10
2
'R-Glyceryl 6-deoxy-6-sulfonato-α-D-glucopyranoside, cyclohexylammonium salt (2'R-2).
Sodium 2'R/S-glyceryl 6-deoxy-6-sulfonato-α-D-glucopyranoside 2 (0.300 g, 0.880 mmol) in water was
+
passed through an Dowex 50 (H form) to obtain an aqueous solution of the sulfonic acid, which was
immediately treated with cyclohexylamine (0.104 g, 1.05 mmol) and evaporated to dryness to yield the
cyclohexylamine salt of the mixture of diastereomers, as a semicrystalline solid. This was fractionally
crystallized from ethanol (3 times) to yield the pure 2'R-stereoisomer (0.050 g, 14% for the combined
30
o
25
30
fractional recrystallizations). mp 190.5-192 °C (lit. 191-193 C); [] +75.3° (c 0.32, CH OH) (lit.
D
3
[]D25 +74.5° (c 18, H O, pH 4)); H NMR (400 MHz, CD OD) δ 4.77 (1 H, d, J = 3.7 Hz, H1), 4.14–
1
2
3
1,2
4
.01 (2 H, m, H5,1'a), 3.93–3.85 (1 H, m, H2'), 3.69–3.51 (3 H, m, H3,3'a,3'b), 3.44–3.32 (3 H, m,
H2,1'b,6a), 3.11–3.00 (2 H, m, H4, cyclohexyl-CH), 2.91 (1 H, dd, J6b,6a = 14.2, J5,6b = 9.3 Hz, H6b),
.06–1.94 (2 H, m, cyclohexyl-CH ), 1.92–1.79 (2 H, m, cyclohexyl-CH ), 1.74–1.64 (1 H, m,
2
2
2
13
1
cyclohexyl-CH ), 1.46–1.12 (5 H, m, cyclohexyl-CH ); C{ H} NMR (101 MHz, CD OD) δ 98.8 (C1),
2
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
3.7 (C3), 73.6 (C4), 72.3 (C2), 71.1 (C2'), 69.4 (C1'), 68.4 (C5), 62.8 (C3'), 52.8 (C6), 50.1 (1 C,
–
cyclohexyl-CH), 30.5, 24.5, 23.9 (5 C, cyclohexyl-CH ); HR-ESI-MS calcd for C H O S [M ]
2
9
17 10
3
17.0542, found 317.0533.
2
. PNPSQ synthesis
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-Nitrophenyl 6-S-benzoyl-6-thio-α-D-glucopyranoside (12). A solution of 4-nitrophenyl -D-
glucopyranoside 11 (0.100 g, 0.332 mmol) and thiobenzoic acid (59 l, 0.50 mmol) in dry THF (2.0
ml) at 0 °C was added to a mixture of PPh (0.13 g, 0.50 mmol) and DIAD (98 l, 0.50 mmol) in THF
3
(3.0 ml) at 0 °C. The reaction mixture was allowed to warm to r.t. and was stirred overnight. The mixture
was concentrated, and the residue was purified by flash chromatography (EtOAc/pet. spirit, 30–100%),
to afford 12 as a white solid (0.114 g, 81%). m.p. 103–105 °C); []D23 +118.4° (c 0.50, CH OH); H
1
3
NMR (500 MHz, CD OD) δ 8.07–8.04 (2 H, m, Ar), 7.78–7.76 (2 H, m, Ph), 7.61–7.57 (1 H, m, Ph),
3
7
.45–7.41 (2 H, m, Ph), 7.27–7.23 (2 H, m, Ar), 5.69 (1 H, d, J1,2 = 3.7 Hz, H1), 3.85 (1 H, dd, J = J
2,3 3,4
1
3
1
9.2 Hz, H3), 3.74–3.68 (3 H, m, H2,5,6b), 3.36–3.34 (1 H, m, H4), 3.07–3.02 (1 H, m, H6a); C{ H}
NMR (101 MHz, CD OD) δ 192.4 (C=O), 162.6, 143.6, 138.0, 134.7, 129.7, 127.9, 126.4, 118.0 (8 C,
3
Ar,Ph), 98.2 (C1), 75.0 (C4), 74.8 (C3), 73.1, 73.0 (2 C, C2,5), 31.6 (C6); HR-ESI-MS calcd for
+
C H NO S [M + H] 422.0904, found 422.0904.
19 19
8
Potassium 4-nitrophenyl 6-deoxy-6-sulfonato-α-D-glucopyranoside (7). Oxone (0.14 g, 0.22 mmol)
was added to a solution of 12 (37.0 mg, 0.088 mmol) and KOAc (72 mg, 0.73 mmol) in glacial AcOH
(3.0 ml). The mixture was stirred at rt for 24 h. The mixture was concentrated and the residue was
purified by flash chromatography (EtOAc/MeOH/H O, 19:2:17:2:1) and C reversed phase
2
18
2
3
chromatography (H O/CH CN, 95:5), affording 7 as a white solid (21.2 mg, 61%). m.p. 163 °C; []
2
3
D
1
+166.2° (c 0.42, CH OH); H NMR (400 MHz, CD OD) δ 8.22 (2 H, d, J 9.1 Hz, Ar), 7.44 (2 H, d, J
3
3
9
.1 Hz, Ar), 5.52 (1 H, d, J1,2 3.6 Hz, H1), 4.16 (1 H, ddd, J = 9.3, J = 8.1, J5,6b = 2.2 Hz, H5), 3.88
4,5 5,6a
(1 H, dd, J3,4 = 9.4, J2,3 = 9.3 Hz, H3), 3.65 (1 H, dd, H2), 3.37 (1 H, dd, J6a,6b = 14.4 Hz, H6b), 3.28 (1
1
3
1
H, dd, H4), 3.02 (1 H, dd, H6a); C{ H} NMR (101 MHz, CD OD) δ 164.1, 144.0, 126.5, 118.9 (4 C,
3
Ar), 100.0 (C1), 74.9 (C4), 74.5 (C3), 73.0 (C2), 71.2 (C5), 54.5 (C6); HR-ESI-MS calcd for
–
C H NO S [M ] 364.0344 found 364.0331.
1
2
14
10
13
3
. Synthesis of SQ and ( C )-SQ
6
Methyl 2,3,4-tri-O-acetyl-6-S-acetyl-6-deoxy-6-thio-α-D-glucopyranoside (14). A mixture of
methy1 2,3,4-tri-O-acetyl-6-deoxy-6-iodo-α-D-glucopyranoside27 13 (6.70 g, 0.016 mol) and KSAc
(4.50 g, 0.039 mol) in acetone (200 ml) was refluxed for 24 h. The reaction mixture was concentrated
using the rotary evaporator, dissolved in ethyl acetate, washed with water and brine and dried (MgSO4).
The mixture was evaporated and purified with flash chromatography (gradient elution, EtOAc/pet.
spirit., 30:70 to 70:30), affording 14 as a yellow oil (5.93 g, 100%). []D23 +94.1° (c 0.75, CHCl ; lit.
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[
] +80.8°); H NMR (500 MHz, CDCl ) δ 5.43 (1 H, dd, J = 9.3, J = 10.1 Hz, H3), 4.93 (1 H, dd,
D
3
3,4
2,3
J = 9.9 Hz, H4), 4.89–4.83 (2 H, m, H1,2), 3.91 (1 H, ddd, J = 7.0, J5,6b = 3.0, J = 10.0 Hz, H5),
4
,5
5,6a
4,5
3
2
.39 (3 H, s, OCH ), 3.20 (1 H, dd, J
= 14.2 Hz, H6b), 3.06 (1 H, dd, H6a), 2.34 (3 H, s, SAc), 2.07,
6a,6b
3
1
3
1
.06, 1.99 (9 H, 3s, 3 x OAc); C{ H} NMR (126 MHz, CDCl ) δ 194.7 (SC=O), 170.3, 170.2, 170.0
3
(
3 C, OC=O), 96.7, 71.1, 71.0 (3 C, C1,2,4), 70.1 (C3), 68.4 (C5), 55.5 (OCH ), 30.6 (SAc), 30.2 (C6),
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
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4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
2
0.9–20.8 (3 C, CH ); HR-ESI-MS calcd for C H O S [M+H] 379.1057, found 379.1056.
3
15 22
9
Potassium methyl 2,3,4-tri-O-acetyl-6-deoxy-6-sulfonato-α-D-glucopyranoside (15). Oxone (21.0
g, 0.0340 mol) was added to a solution of 14 (5.10 g, 0.0130 mol) and NaOAc (18.0 g, 0.130 mol) in
glacial AcOH (40 ml). The mixture was stirred at rt for 24 h. The mixture was concentrated and the
residue was purified by flash chromatography (EtOAc/MeOH/H O, 15:2:15:2:1), affording 15 as a
2
3
4
23
34
1
white solid (5.36 g, 94%). m.p. 152 °C (lit. 113 °C); [] +100.4° (c 1.31, CHCl ; lit. +129.3°); H
D
3
NMR (499 MHz, CDCl ) δ 5.36 (1 H, t, J = J 9.4 Hz, H3), 4.97 (2 H, br. s, H1,4), 4.87 (1 H, d, H2),
3
2,3
3,4
4
2
1
.23 (1 H, br. s, H5), 3.40 (3 H, s, OCH ), 3.21 (1 H, br. s, H6b), 3.11 (1 H, d, J5,6a = 8.1 Hz, H6a),
3
1
3
1
.05–1.95 (6 H, br. s, 2 x Ac), 1.91 (3 H, s, Ac); C{ H} NMR (101 MHz, CDCl ) δ 171.0, 170.6,
3
70.1 (3 C, C=O), 96.6, 71.1 (2 C, C1,4), 70.8 (C2), 70.5 (C3), 65.9 (C5), 55.9 (OCH ), 51.0 (C6), 21.0,
3
–
20.9, 20.8 (3 C, CH ); HR-ESI-MS calcd for C H O S [M ] 383.0654 found 383.0664.
3
13 19 11
Potassium methyl 6-deoxy-6-sulfonato-α-D-glucopyranoside (16). Potassium methyl 2,3,4-tri-O-
acetyl-6-deoxy-6-sulfonato-α-D-glucopyranoside 15 (5.30 g, 0.0130 mol) was dissolved in MeOH and
treated with Na (50 mg) and stirred at rt for 2 h. The mixture was neutralized with resin and purified by
flash chromatography (EtOAc/MeOH/H O/, 10:2:15:2:1), affording 16 as a low-melting,
2
hygroscopic white solid (3.63 g, 98%). []D23 +87.1° (c 0.90, H O; lit. [] +82.1° in MeOH); H
35
22
1
2
D
NMR (500 MHz, D O) δ 4.80–4.78 (1 H, br. s, H1), 4.04 (1 H, dt, J = J = 1.3, J5,6a = 9.9 Hz, H5),
2
4,5
5,6b
3.68 (1 H, dd, J = 8.0, J = 10.8 Hz, H3), 3.60 (1 H, dd, J = 3.8 Hz, H2), 3.48 (3 H, s, OCH ), 3.41
2
,3
3,4
1,2
3
1
3
1
(
1 H, dd, J6b,6a = 14.7 Hz, H6b), 3.27 (1 H, dd, H4), 3.09 (1 H, dd, H6a); C{ H} NMR (101 MHz,
D O) δ 98.9 (C1), 72.9 (C3), 72.3 (C4), 71.1 (C2), 67.7 (C5), 55.1 (OCH ), 52.0 (C6); HR-ESI-MS
2
3
–
calcd for C H O S[M ] 257.0337, found 257.0319.
7
13
8
Potassium 6-deoxy-6-sulfonato-D-glucopyranose (1). Compound 16 (2.40 g, 8.60 mmol) was
dissolved in 2 M HCl and stirred at 100 °C for 24 h. The mixture was concentrated under reduced
pressure and the residue was purified by flash chromatography (EtOAc/MeOH/H O, 10:2:12:2:1),
2
affording 1 as a low-melting, hygroscopic light yellow solid (2.29 g, 100%). []D23 +42.1° (c 0.64, H O;
2
3
6
20
1
lit. []
+71.7°); H NMR (400 MHz, D O) δ 5.22 (1 H, d, J 3.7 Hz, H1α), 4.68 (1 H, d, J 8.0
2 1,2 1,2
5
46
Hz, H1β), 4.24 (1 H, t, J4,5 = J = 9.4 Hz, H5α), 3.80 (1 H, t, J = J = 9.2 Hz, H5β), 3.73 (1 H, t, J
2,3
5,6
4,5
5,6
=
J = 9.4 Hz, H3α), 3.56 (1 H, dd, H2α), 3.50 (1 H, dd, J = 5.9, J = 15.3 Hz, H3β), 3.41 (1 H, d,
3,4 2,3 3,4
13
1
J6b,6a = 14.7 Hz, H6bβ), 3.35–3.25 (3 H, m, H4α,2β,4β), 3.13–3.04 (3 H, m, H6bα,6aα,6aβ); C{ H}
NMR (101 MHz, D O) δ 95.9 (C1β), 91.9 (C1α), 75.5 (C3β), 74.0 (C2β), 72.6 (C4α), 72.5 (C3α), 72.3
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5
5
6
(
C4β), 72.1 (C5β), 71.3 (C2α), 67.7 (C5α), 52.22, 52.16 (2 C, C6α,6β); HR-ESI-MS calcd for C H O S
6
11
8
–
[M ] 243.0180, found 243.0202.
1
3
Methyl D-( C )glucopyranoside (17). Acetyl chloride (0.80 mL, 11.2 mmol) was added to a solution
6
1
3
of D-( C )glucose (2.93 g, 15.8 mmol) in MeOH (60 mL). The solution was refluxed for 16 h and
6
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
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0
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9
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7
8
9
0
1
2
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5
6
7
8
9
0
evaporated to dryness under reduced pressure to afford the title compound as a mixture of anomers
1
(2.5:1, α:β) as a colourless glass. This was not purified, but was used directly in the next step. H NMR
1
= 173.2 Hz, H1-α), 4.39 (1 H, dd, 1JC1,H1 = 166.0, JH1,H2 = 7.7
C1,H1
(400 MHz, D O) δ 4.80 (1 H, dd, J
2
Hz, H1-β), 4.14–3.80 (2 H, m, H6a-α,6a-β), 3.97–3.69 (2 H, m, H6b-α,6b-β), 3.86–3.55 (1 H, m, H2-
α), 3.78–3.45 (2 H, m, H3-α,5-α), 3.58 (3 H, d, 3JC1,H-Me = 4.3 Hz, OMe-β), 3.69–3.38 (2 H, m, H3-β,5-
β), 3.42 (3 H, d, 3JC1,H-Me = 3.9 Hz, OMe-α), 3.41–3.17 (2 H, m, H4-α,4-β), 3.33–3.01 (1 H, m, H2-β);
1
3
C{ H} NMR (101 MHz, D O) δ 103.1 (1 C, d, J = 46.5 Hz, C1-β), 99.2 (1 C, dd, 1J1,2 = 46.0, 2J1,3
1
1
2
1,2
=
9.2 Hz, C1-α), 76.9–74.9 (2 C, m, C3-β,5-β), 73.7–72.2 (2 C, m, C2-β,3-α), 72.1–70.4 (2 C, m, C5-
α,2-α), 70.3–68.8 (2 C, m, C4-α,4-β), 61.3–59.4 (2 C, m, C6-α,6-β), 55.0–54.8 (2 C, m, OMe-α,OMe-
1
3
12
+
β); HR-ESI-MS calcd for C CH O Na [M + Na] 223.0889, found 223.0883.
6
14
6
1
3
Methyl 2,3,4-tri-O-acetyl-6-deoxy-6-iodo-D-( C )glucopyranoside (18). A solution of 1 (3.15 g,
6
1
5.8 mmol), PPh (6.26 g, 23.9 mmol), I (5.65 g, 22.3 mmol) and imidazole (3.30 g, 48.5 mmol) in
3 2
toluene (75 mL) was heated at 75°C for 2.5 h with vigorous stirring. The mixture was cooled, H O (90
2
mL) was added and the mixture was stirred vigorously for 15 min. The toluene phase was extracted
with H O (3 × 30 mL), and the combined aqueous phase was washed with toluene (30 mL) and
2
evaporated to dryness under reduced pressure. Ac O (33 mL) and pyridine (67 mL) were added to the
2
residue and the mixture was stirred at rt for 16 h and then evaporated under reduced pressure. The
residue was partitioned between toluene (40 mL) and H O (30 mL). The aqueous phase was washed
2
with toluene (3 × 20 mL). The combined toluene phase was dried (MgSO ) and evaporated to dryness
4
under reduced pressure The residue was crystallized from hot EtOH (26 mL) to afford 18 as a mixture
.
of anomers (10:1, α:β) as colourless crystals (2.30 g, 34%). m.p. 137-139°C; [α] +103.3 (c 0.575,
D
1
CHCl ); H NMR (400 MHz, CDCl ) δ 5.72–5.24 (1 H, m, H3-α), 5.20–4.61 (3 H, m, H1-α,2-α,4-α),
3
3
4
.02–3.57 (1 H, m, H5-α), 3.48 (3 H, d, 3JC1,H-Me = 4.5 Hz, OMe-α), 3.41 (3 H, d, 3JC1,H-Me = 4.4 Hz,
OMe-β), 3.52–3.07 (1 H, m, H6a-α), 3.36–2.90 (1 H, m, H6b-α), 2.10–2.06 (6 H, m, 2 × Ac-β), 2.09–
1
3
1
2.04 (6 H, m, 2 × Ac-α), 2.02 (3 H, s, Ac-β), 2.00 (3 H, s, Ac-α); C{ H} NMR (101 MHz, CDCl ) δ
3
1
70.5–169.4 (3 C, m, C=O-α), 101.6 (1 C, dd, 1J1,2 = 46.9, 2J1,3 = 25.8 Hz, C1-β), 96.9 (1 C, d, 1J1,2
=
1
1
45.4 Hz, C1-α), 74.4–73.2 (1 C, m, C4-β), 72.6 (1 C, t, J = J = 39.7 Hz, C4-α), 72.1–71.7 (1 C, m,
3
,4
4,5
C2-β), 71.1 (1 C, dd, 1J1,2 = 45.0, 1J2,3 = 41.5 Hz, C2-α), 69.7 (1 C, t, J = J = 40.0 Hz, C3-α), 68.7
1
1
2,3
3,4
1
1
1
1
1
1
(
1 C, t, J = J = 41.5 Hz, C5-α), 67.2 (1 C, t, J = J = 42.7 Hz, C3-β), 62.0 (1 C, d, J = J
4,5 5,6 3,4 4,5 4,5 5,6
=
44.2 Hz, C5-β), 55.9 (1 C, s, OMe-α), 20.9 (3 C, s, COMe-α), 3.8 (1 C, d, 1J5,6 = 42.7 Hz, C6-α), 3.1
1 C, d, 1J5,6 = 40.8 Hz, C6-β); HR-ESI-MS calcd for C C H O INa [M + Na] 459.0224, found
13
12
+
(
6 7 19 8
459.0216.
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Methyl 2,3,4-tri-O-acetyl-6-S-acetyl-6-deoxy-6-thio-D-( C )glucopyranoside (19). A solution of 2
6
(2.30 g, 5.3 mmol) and KSAc (1.53 g, 13.4 mmol) in acetone (80 mL) was refluxed for 16 h. The
mixture was evaporated to dryness under reduced pressure and dissolved in EtOAc (42 mL). The
organic phase was washed with H O (3 × 11 mL) and saturated NaHCO solution (3 × 11 mL), dried
2
3
(
MgSO ) and evaporated to dryness under reduced pressure. The residue was purified by flash
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
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5
5
5
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0
1
2
3
4
5
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0
1
2
3
4
5
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7
8
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0
1
2
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4
5
6
7
8
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chromatography (EtOAc/pet. spirit, 30:70 → 70:30) to afford 19 as a mixture of anomers (20:1, α:β) as
1
a reddish-yellow oil (1.68 g, 83%). [α] +90.2 (c 1.04, CHCl ); H NMR (600 MHz, CDCl ) δ 5.63–
D
3
3
.25 (1 H, m, H3-α), 5.12–4.66 (3 H, m, H4-α,1-α,2-α), 4.06–3.75 (1 H, m, H5-α), 3.48 (3 H, d, 3JC1,H-
5
= 4.7 Hz, OMe-β), 3.38 (3 H, d, 3JC1,H-Me = 4.6 Hz, OMe-α), 3.33–3.04 (1 H, m, H6a-α), 3.21–2.91
Me
1
3
1
(
1 H, m, H6b-α), 2.33 (3 H, s, SAc-α), 2.10–2.05 (6 H, m, 2 × OAc-α), 1.98 (3 H, s, OAc-α); C{ H}
1
NMR (151 MHz, CDCl ) δ 194.8 (1 C, s, SC=O-α), 172.2–169.6 (3 C, m, OC=O-α), 101.5 (1 C, d, J
3
1,2
=
48.6 Hz, C1-β), 96.7 (1 C, d, 1J1,2 = 46.6 Hz, C1-α), 73.6–72.5 (2 C, m, C2-β,4-β), 71.6–69.5 (3 C, m,
1
1
1
1
C2-α,3-α,4-α), 68.3 (1 C, t, J = J = 41.2 Hz, C5-α), 67.2 (1 C, t, J = J = 42.8 Hz, C3-β), 62.1
4
,5
5,6
2,3
3,4
1
1
(
1 C, d, J = J = 44.5 Hz, C5-β), 55.5 (1 C, s, OMe-α), 30.6 (1 C, s, SAc-α), 30.5–30.4 (1 C, m, C6-
4,5 5,6
β), 30.2 (1 C, d, 1J5,6 = 42.0 Hz, C6-α); HR-ESI-MS calcd for C C H O SNa [M + Na] 407.1084,
13
12
+
6
9
22
9
found 407.1077.
13
Potassium methyl 2,3,4-tri-O-acetyl-6-deoxy-6-sulfonato-D-( C )glucopyranoside (20). Oxone
6
(
5.53 g, 9.0 mmol) was added to a mixture of 3 (1.68 g, 4.4 mmol) and KOAc (4.32 g, 44.0 mmol) in
AcOH (17 mL) and H O (2.4 mL). The mixture was stirred at rt for 20 h, then evaporated to dryness
2
under reduced pressure. The residue was purified by flash chromatography (EtOAc/MeOH/H O, 12:2:1
2
→
5:2:1) to afford 20 as a mixture of anomers (25:1, α:β) as a colourless solid (1.57 g, 84%). [α]D
1
1
+
108.1° (c 1.06, CHCl ); H NMR (600 MHz, CDCl ) δ 5.44 (1 H, d, J
= 150.2 Hz, H3-α), 5.39–
3
3
C3,H3
4
.70 (3 H, m, H1-α,4-α,2-α), 4.30 (1 H, d, 1JC5,H5 = 148.7 Hz, H5-α), 3.59 (3 H, s, OMe-β), 3.47 (3 H,
s, OMe-α), 3.40–2.95 (2 H, m, H6a-α, H6b-α), 2.09–2.03 (6 H, m, 2 × OAc-α), 1.99 (3 H, s, OAc-α);
1
3
1
1
C{ H} NMR (101 MHz, CDCl ) δ 170.9, 170.5, 170.1 (3 C, 3 x s, C=O-α), 102.1 (1 C, d, J = 42.5
3
1,2
Hz, C1-β), 96.5 (1 C, d, 1J1,2 = 41.6 Hz, C1-α), 72.1–69.6 (3 C, m, C4-α,3-α,2-α), 65.9 (1 C, t, J =
1
4,5
1J5,6 = 40.1 Hz, C5-α), 55.7 (1 C, s, OMe-α), 51.2 (1 C, d, 1J5,6 = 40.1 Hz, C6-α), 21.0–20.7 (3 C, m,
1
3
12
–
O=CMe-α); HR-ESI-MS calcd for C C H O S [M ] 389.0849, found 389.0863.
6
7
19 11
1
3
Potassium methyl 6-deoxy-6-sulfonato-D-( C )-glucopyranoside (21). A mixture of 4 (1.57 g, 3.7
6
mmol) and K CO (0.37 g, 2.7 mmol) in MeOH (12 mL) was stirred at rt for 4 h. The mixture was
2
3
neutralized with AcOH (0.20 mL, 3.5 mmol) and evaporated to dryness under reduced pressure. The
residue was purified by flash chromatography (EtOAc/MeOH/H O, 6:2:1 → 3:2:1) to afford 21 as a
2
1
mixture of anomers (30:1, α:β) as a colourless solid (0.99 g, 89%). [α] +85.7° (c 0.82, MeOH); H
D
NMR (400 MHz, D O) δ 4.99–4.46 (1 H, m, H1-α), 4.22–3.75 (1 H, m, H5-α), 3.85–3.46 (1 H, m, H3-
2
α), 3.75–3.39 (1 H, m, H2-α), 3.42 (3 H, d, 3JC1,H-Me = 4.2 Hz, OMe-α), 3.55–3.14 (1 H, m, H6a-α),
1
3
1
3.44–2.94 (1 H, m, H4-α), 3.26–2.80 (1 H, m, H6b-α); C{ H} NMR (101 MHz, D O) δ 102.9 (1 C, d,
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1J1,2 = 41.9 Hz, C1-β), 98.8 (1 C, dd, J = 45.8, J = 6.9 Hz, C1-α), 73.6–71.6 (2 C, m, C3-α,2-α),
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1
1
71.0 (1 C, dd, J = 44.3, J = 38.3 Hz, C4-α), 67.6 (1 C, dd, J = 41.3, J = 35.7 Hz, C5-α), 55.0
3
,4
4,5
5,6
4,5
(
1 C, s, OMe-α), 51.9 (1 C, d, 1J5,6 = 41.5 Hz, C6-α); HR-ESI-MS calcd for 13C CH O S [M ]
12
–
6 13 8
263.0532, found 263.0521.
0
1
2
3
4
5
6
7
8
9
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13
Potassium 6-deoxy-6-sulfonato-D-( C )glucose (22). Aq. HCl (1.6 mL, 2 M, 3.1 mmol) was added
6
to a solution of 5 (0.97 g, 3.2 mmol) in H O (8 mL). The mixture was heated at 100°C for 24 h and
2
evaporated to dryness under reduced pressure. The residue was purified by reverse phase
+
chromatography (100% H O) and treated with Dowex 50 ion exchange resin (K form) to afford 22 as
2
1
a mixture of anomers (1:1, α:β) as a pale yellow solid (0.68 g, 73%). [α] +26.8° (c 1.30, H O); H NMR
D
2
1
= 171.5 Hz, H1-α), 4.67 (1 H, dd, 1JC1,H1 = 161.5, JH1,H2 = 7.8
(
400 MHz, D O) δ 5.21 (1 H, d, J
2
C1,H1
Hz, H1-β), 4.44–4.02 (1 H, m, H5-α), 4.02–3.28 (4 H, m, H5-β,3-α,2-α,3-β), 3.41 (2 H, dd, 1JC6,H6
=
1
34.7, 2JH6,H6’ = 14.5 Hz, H6a-β,6a-α), 3.49–3.06 (3 H, m, H6b-β,6b-α,4-β), 3.30–2.86 (2 H, m, H4-α,2-
β); C{ H} NMR (101 MHz, D O) δ 95.8 (1 C, dd, J = 45.1, 2J1,3 = 4.8 Hz, C1-β), 91.9 (1 C, dd,
1
3
1
1
2
1,2
1J1,2 = 44.5, 2J1,3 = 13.5 Hz, C1-α), 76.1–74.9 (1 C, m, C3-β), 74.6–73.3 (1 C, m, C2-β), 73.1–71.9 (4
C, m, C3-α,4-β,4-α,5-β), 71.8–70.6 (1 C, m, C2-α), 68.4–66.9 (1 C, m, C5-α), 53.2–51.5 (2 C, m, C6-
1
3
–
α,6-β); HR-ESI-MS calcd for C H O S [M ] 249.0376, found 249.0363.
6
11
8
4. Synthesis of SF
Methyl 1,3-O-isopropylidene-α-D-fructofuranoside (23). A mixture of fructose (1.0 g, 5.6 mmol)
and TsOH (48 mg, 0.28 mmol) in anhydrous MeOH (5.6 ml) was stirred overnight. The reaction mixture
was treated with 2,2-dimethoxypropane (5.6 ml) and stirred at rt for 45 min. Anhydrous NaHCO to
3
neutralize the solution, and the mixture was filtered, evaporated and the residue was purified by flash
chromatography (CHCl /MeOH/Et N, 100:0:580:20:5) to afford 23 as a yellow oil (0.832 g, 64%).
3
3
[
]D23 +34.5° (c 0.50, CHCl ; lit. [] +38.6° in CHCl ); H NMR (500 MHz, CDCl ) δ 4.11 (1 H, dt,
28
1
3
D
3
3
J = 3.3, J = 4.9 Hz, H5), 4.04–3.89 (4 H, m, H1b,1a,3,4), 3.84–3.75 (2 H, m, H6b,6a), 3.30 (3 H, s,
5,6
4,5
1
3
1
OCH ), 1.45, 1.36 (6 H, 2s, 2 x CH ); C{ H} NMR (126 MHz, CDCl ) δ 101.4, 98.9 (2 C, C2,7), 87.7
3
3
3
(C5), 79.8, 77.8, 61.9 (3 C, C1,3,4), 62.9 (C6), 48.8 (OCH ), 27.9, 19.4 (2 C, CH ); HR-ESI-MS calcd
3
3
+
for C H O [M+H] 235.1176, found 235.1176.
1
0
18
6
Methyl 6-S-benzoyl-1,3-O-isopropylidene-6-thio-α-D-fructofuranoside (24). A solution of 23 (0.40
g, 1.7 mmol) and thiobenzoic acid (0.30 ml, 2.6 mmol) in dry THF (10 ml) at 0 °C was added to a
mixture of PPh (0.67 g, 2.6 mmol) and DIAD (0.50 ml, 2.6 mmol) in THF (10 ml) at 0 °C. The reaction
3
mixture was allowed to warm to r.t. and stirred for 1.5 h. The mixture was concentrated and the residue
was purified by flash chromatography (EtOAc/pet. spirit/Et N, 1:9:0.13:7:0.1), to afford 24 as a
3
yellow oil (0.550 g, 91%). []D23 +43.4° (c 0.62, CHCl ); H NMR (500 MHz, CDCl ) δ 7.93-7.89 (2
1
3
3
H, m, Ph), 7.51–7.46 (1 H, m, Ph), 7.39–7.33 (2 H, m, Ph), 4.11 (1 H, dt, J = 3.2, J = 6.5 Hz, H5),
5
,6
4,5
3
.99–3.82 (4 H, m, H1a,1b,3,4), 3.41–3.34 (2 H, m, H6b,6a), 3.22 (3 H, s, OCH ), 1.37, 1.33 (6 H, 2s,
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x CH ); C{ H} NMR (126 MHz, CDCl ) δ 191.2 (C=O), 136.6, 133.4, 128.5, 127.2 (4 C, Ph), 102.3,
3 3
98.7 (2 C, C2,7), 85.1 (C5), 80.3, 79.8, 61.6 (3 C, C1,3,4), 48.5 (OCH ), 31.6 (C6), 27.4, 19.9 (2 C,
3
+
CH ); HR-ESI-MS calcd for C H O S [M+H] 355.1201, found 355.1209.
3
17 22
6
Potassium methyl 6-deoxy-1,3-O-isopropylidene-6-sulfonato-α-D-fructofuranoside (25). Oxone
2.4 g, 3.9 mmol) was added to a solution of 24 (0.55 g, 1.6 mmol) and KOAc (1.5 g, 15 mmol) in
1
1
1
1
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0
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8
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0
(
glacial AcOH (8.0 ml) and the mixture was stirred at rt for 24 h. The mixture was concentrated and the
residue was purified by flash chromatography (EtOAc/MeOH/H O, 10:2:17:2:1), affording 25 as a
2
white crystalline solid (0.470 g, 90%). m.p. 177 °C; []D23 +21.2° (c 0.55, MeOH); H NMR (500 MHz,
1
CD OD) δ 4.36–4.30 (1 H, m, H5), 4.09 (1 H, dd, J =1.2, J = 4.6 Hz, H4), 4.04 (1 H, d, J = 1.3 Hz,
3
3,4
4,5
3,4
H3), 3.98 (1 H, d, J1a,1b = 12.2 Hz, H1b), 3.82 (1 H, d, H1a), 3.35 (3 H, s, OCH ), 3.28–3.18 (2 H, m,
3
1
3
1
H6b,6a), 1.47 (3 H, s, CH ), 1.38 (3 H, s, CH ); C{ H} NMR (126 MHz, CD OD) δ 103.9, 100.4 (2
3
3
3
C, C2,7), 82.8 (C3), 82.0, 81.9 (2 C, C4,5), 62.8 (C1), 55.8 (C6), 48.8 (OCH ), 27.1, 21.2 (2 C, CH );
3
3
–
HR-ESI-MS calcd for C H O S [M ] 297.0650, found 297.0656.
1
0
17
8
Potassium 6-deoxy-6-sulfonato-D-fructofuranose (3). Methyl 6-deoxy-1,3-O-isopropylidene-6-
sulfonato-α-D-fructofuranoside potassium salt (0.13 g, 0.39 mmol) was dissolved in 1:1 acetic acid/H O
2
(
3 mL) and stirred at 25–30 °C for 72 h. The mixture was concentrated to afford 3 (3.7:1 : ratio of
anomers) as a colourless oil (0.107 g, 100%). []D23 +13.9° (c 0.86, H O); H NMR (500 MHz, D O) δ
1
2
2
4.35 (1 H, ddd, J = 4.8, J = 7.1, J5,6b = 8.1 Hz, H5-β), 4.21–4.09 (4 H, m, H3-β,3-α,4-α,5-α), 4.00
4
,5
5,6a
(
1 H, dd, J4,5 = 5.4, J = 6.9 Hz, H4-β), 3.70 (1H, d, J
= 12.1 Hz, H1bβ) 3.63 (1 H, d, J1b,1a = 12.1
3,4
1b,1a
Hz, H1aβ), 3.60 (1 H, d, J1b,1a = 12.2 Hz, H1bα), 3.57 (1 H, d, J1b,1a = 12.2 Hz, H1aα), 3.37–3.20 (4 H,
1
3
1
m, H6); C{ H} NMR (101 MHz, D O) δ 104.5 (C2-β), 101.7 (C2-α), 81.5 (C3-β), 79.5 (C4-β), 77.8,
2
7
6.1, 74.6 (3 C, C3-α,4-α,5-α), 76.7 (C5-β), 62.8 (C1-β), 62.7 (C1-α), 55.2 (C6-α), 53.7 (C6-β); HR-
–
ESI-MS calcd for C H O S [M ] 243.0180, found 243.0171.
6
11
8
5. Synthesis of SFP
Sulfofructose-1-phosphate, bis(triethylammonium) salt (4). A solution was prepared containing 3
(1000 µL of 172 mM stock; 0.048 g, 0.172 mmol), Na ATP (800 µL of 442 mM stock, 0.252 g, 0.353
3
mmol), Tris-buffer pH 7.5 (800 µL of 1.00 M stock), MgCl (600 µL of 2000 mM stock), deionized
2
water (Milli-Q; 775 µL). The final volume was 4.0 mL; the final concentrations were: SF (3) 43 mM,
ATP 88 mM, Tris-buffer pH 7.5 100 mM and MgCl 150 mM. YihV (12 mg/mL 25 µL) was added to
2
the solution to start the reaction, which was kept at rt for 72 h. When the reaction was completed the
solution was heated at 80 °C for 3 min. The supernatant was filtered through a 0.22 micron filter (Merck
Millipore Ltd.). The reaction mixture was purified by HPLC-MS on a Shimadzu LCMS-2020 equipped
with an LC-30AD pump, SIL-30AC autosampler and FRC-10A fraction collector, using a 150 x 21.2
mm ACE 10 SuperC18 column (part no. ACE-1311-1520). Solvent A: 50 mM aqueous
triethylammonium bicarbonate pH 8.2; Solvent B: 50 mM triethylammonium bicarbonate in methanol.
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The solvent program was 0-40% solvent B over 26 minutes, 40-100% solvent B over 0.5 minutes, 100%
solvent B for 5.5 minutes and finally 0% solvent B for 8 minutes, flow rate of 8 mL/min. Fractions
containing the product were combined and lyophilized to afford 4 (4:1 : ratio of anomers) as a
colourless glass (49 mg, 54%). []D23 +0.23° (c 3.65, H O); H NMR (600 MHz, D O) δ 4.36 (0.22 H,
1
2
2
ddd, J = 7.4, 6.3, 4.7 Hz, H5-β), 4.18–4.09 (2.74 H, m, H3,4-α,5-α), 4.05 (0.22 H, dd, J4,5 = 6.2, J3,4
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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0
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.1 Hz, H4-β), 3.95–3.80 (2 H, m, H1a-α,1a-β,1b-α,1b-β), 3.33–3.31 (2 H, m, H6a-α,6a-β,6b-α,6b-β),
3.26 (1 H, td, J = 7.9, 5.9 Hz, H6b), 3.21 (12 H, q, J = 7.3 Hz, CH CH ), 1.29 (18 H, t, J = 7.3 Hz,
3
2
CH ); C{ H} NMR (151 MHz, D O) δ 104.1 ( J = 9.2 Hz, C2-β), 100.8 (d, 3JC,P = 9.8 Hz), 81.1,
1
3
1
3
3
2
C,P
7
9.2 (C3-β,4-β), 77.9 (C3-α,4-α,5-α), 77.7 (C5-β), 76.3, 75.1 (C3- α,4-α,5-α), 65.8 (C1-α), 65.3 (C1-β),
3
1
1
5
5.3 (C6-α), 54.0 (C6-β), 46.6 (CH CH ), 8.1 (CH ); P{ H} NMR (202 MHz, D O) δ 0.70; HR-ESI-
3
2
3
2
+
MS calcd for C H O PSNa [M+Na ] 346.9808 found 346.9808.
6
13 11
6
. Synthesis of SLA
Sodium DL-sulfolactaldehyde diethyl acetal (27). A solution of DL-glycidaldehyde diethylacetal 26
0.100 g, 0.680 mmol) and anhydrous sodium sulfite (0.430 g, 3.40 mmol) in water (5 mL) was heated
(
under reflux under nitrogen for 18 h. The solvent was evaporated under reduced pressure to afford a
white solid that was purified by flash chromatography (27:2:1 EtOAc/MeOH/H O + 2% Et N to 15:8:4
2
3
EtOAc/MeOH/H O + 2% Et N) to give the product as the triethylammonium salt (0.150 g). This
2
3
+
material was passed through a column of Dowex 50 (Na form; 9 x 1.5 cm), and the eluent was
1
evaporated to dryness to yield the sodium salt 27 as a glassy film (0.120 g, 71%). H NMR (600 MHz,
CD OD) δ 4.44 (1 H, d, J = 4.7 Hz, H1), 4.10–4.06 (1 H, m, H2), 3.76–3.69 (2 H, m, H1'b,1''b), 3.64–
3
1,2
3
.56 (2 H, m, H1'a,1''a), 3.10 (1 H, dd, J = 14.2, 2.3 Hz, H3a), 2.89 (1 H, dd, J = 14.2, 9.5 Hz, H3b),
1
3
1
1.20 (3 H, m, J = 6.9 Hz, CH ), 1.19 (3 H, t, J = 6.9 Hz, CH ); C{ H} NMR (151 MHz, CD OD) δ
3
3
3
1
03.6 (C1), 68.6 (C2), 63.6 (C1''), 63.0 (C1'), 52.3 (C3), 14.2 (CH ); HR-ESI-MS calcd for C H O S
3 7 15 6
–
[
M ] 227.0589, found 227.0572.
+
DL-Sulfolactaldehyde hydrate, free acid (5). Dowex 50 resin (H form, 150 mg) was added to a
solution of 27 (0.013 g, 0.050 mmol) in D O (0.50 ml). The mixture was stirred at 80 °C for 10 min and
2
was filtered to remove the resin. NMR analysis indicated complete removal of acetal groups affording
5
as the hydrate in D O, along with 2 equivalents of EtOH. The product was stable as a solution at room
2
1
temperature for several days, but decomposed upon removal of solvent. H NMR (400 MHz, D O) δ
2
4
.95 (1 H, d, J1,2 4.3 Hz, H1), 4.01–3.91 (1 H, m, H2), 3.63 (4 H, q, J 7.1 Hz, CH of 2 × EtOH), 3.20
2
(1 H, dd, J2,3b = 2.7, J3a,3b = 14.5 Hz, H3b), 2.98 (1 H, dd, J2,3a = 8.9 Hz, H3a), 1.15 (6 H, t, J 7.1 Hz,
1
3
1
CH of 2 × EtOH); C{ H} NMR (101 MHz, D O) δ 90.6 (C1), 70.2 (C2), 52.6 (C3); HR-ESI-MS
3
2
–
calcd for C H O S [M ] 170.9969, found 170.9974.
3
7
6
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. Synthesis of DHPS
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Cyclohexylammonium 2,3-dihydroxypropanesulfonate (6).
(a) From allyl alcohol: A mixture of allyl alcohol (3.5 mL, 52 mmol), Ac O (15 mL, 159 mmol) and
2
AcOH (5 mL, 88 mmol) was treated dropwise with H SO (96%, 3 mL, 56 mmol). The mixture was
2
4
heated for 3 h at 80°C. The solvent (approx 20 mL) was evaporated under reduced pressure and the
1
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residue was azeotroped with H O (12 × 15 mL) until the distillate’s pH was 6. The residue was dissolved
2
in EtOH (16 mL) and neutralized by addition of cyclohexylamine (7 mL, 61 mmol), at which point the
solution was basic. The solution was evaporated to dryness under reduced pressure. The crude
cyclohexylammonium DHPS was crystallized from EtOH/toluene (14 mL/34 mL) to afford 6 as white,
powdery crystals (5.3 g, 40% over 2 crops). m.p. 52-53 °C; 1H NMR (400 MHz, D O) δ 4.09–3.99 (1
2
H, m, H2), 3.58 (1 H, dd, J3a,3b = 11.8, J2,3b = 3.6 Hz, H3b), 3.47 (1 H, dd, J3a,3b = 11.8, J2,3a = 6.4 Hz,
H3a), 3.07–3.00 (1 H, m, cyclohexyl-CH), 3.00 (1 H, dd, J1a,1b = 14.6, J1b,2 = 4.3 Hz, H1b), 2.91 (1 H,
13
1
dd, J1a,1b = 14.4, J1a,2 = 7.5 Hz, H1a), 1.90–0.97 (10 H, m, cyclohexyl-CH ); C{ H} NMR (101 MHz,
2
D O) δ 68.0 (C2), 64.5 (C3), 53.7 (C1), 50.3 ( cyclohexyl-CH ), 30.3, 24.2, 23.7 (cyclohexyl-CH );
2
2
2
–
HR-ESI-MS calcd for C H O S [M ] 155.0014, found 155.0008.
3
7
5
(b) From glycidol: Anhydrous sodium sulfite (8.50 g, 67 mmol) was added to a stirred solution of
glycidol (1.00 g, 13 mmol) in water (50 mL) and the solution was heated under nitrogen at reflux for
24 hours. After cooling to room temperature the reaction mixture was evaporated under reduced
pressure. The resulting white solid was slurried in methanol (50 mL) for 4 hours, filtered and evaporated
to give crude sodium 2,3-dihydroxypropanesulfonate (2.2 g). This was passed through Dowex 50 (H+
form) and evaporated to obtain crude 2,3-dihydroxypropanesulfonic acid. This was dissolved in water
(
50 mL) again and treated with cyclohexylamine (1.41 g, 14.3 mmol) and followed by evaporating to
dryness. The resulting slightly coloured viscous oil was purified by flash chromatography (30:2:1 to
:2:1 EtOAc/MeOH/H O) to obtain 6 as a viscous oil (1.90 g, 58%). This can be recrystallized from 2-
5
2
propanol/diethyl ether. m.p. 51-52 °C.
Cloning, expression and purification of SF kinase YihV. Escherichia coli yihV (NC_000913.3) was
amplified through colony PCR using ‘TOP10’ E. coli cells (ThermoFisher) as template with the primers
5
’-ATATACATATGATTCGTGTTGCTTGTGTAGG-3’
and
5’-
TGGTGCTCGAGTACAAAAAGTGACAAAAAAGATCG-3’. The product was cloned into
pET21b(+) (Novagen) using the NdeI and XhoI restriction sites to provide pET21b-YihV. This plasmid
was transformed into chemically competent ‘T7 Express’ E. coli cells (NEB), plated onto LB-agar (100
μg/ml ampicillin) and incubated at 37 °C for 16 h. A single colony was used to inoculate 10 ml of LB
media (100 μg/ml ampicillin), which was incubated at 37 °C for 16 h with shaking (180 rpm). This
culture was used to inoculate 1000 ml of S-broth media (100 μg/ml ampicillin), which was incubated
at 37 °C with shaking (250 rpm). When the culture reached an OD600 of 0.7, it was cooled to room
temperature and isopropyl β-D-thiogalactopyranoside added to a final concentration of 100 μM. This
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culture was incubated at 18 °C for 19 h with shaking (220 rpm). The cells were harvested by
centrifugation at 8000 g for 20 min at 4 °C then resuspended in 40 ml of binding buffer (50 mM NaPi,
5
00 mM NaCl, 5 mM imidazole, pH 7.4) containing protease inhibitor (Roche complete EDTA-free
protease inhibitor cocktail) and lysozyme (0.1 mg/ml) by nutating at 4 °C for 30 min. Benzonase (1 μl)
was added to the mixture and cell lysis achieved using sonication. The lysate was clarified by
centrifugation (18,000 g for 20 min at 4 °C) and the supernatant filtered (0.45 μm) before loading onto
a 1 ml HiTrap TALON column (GE Healthcare). The column was washed with 15 ml binding buffer
then YihV was eluted using elution buffer (50 mM NaPi, 500 mM NaCl, 500 mM imidazole, pH 7.4).
Fractions containing YihV (as determined by SDS-PAGE) were further purified by size exclusion
chromatography on a HiLoad 16/600 Superdex 200 column using 50 mM NaPi, 150 mM NaCl, pH 7.4.
Protein purity was estimated at >95% by Image Lab (Bio-Rad) analysis of Coomassie-stained SDS-
PAGE and concentration determined by absorbance at 280 nm. The yield of YihV was 10 mg per litre
of culture.
0
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Single crystal X-ray structure determination of cyclohexylammonium 2’R-glyceryl a-D-
37
sulfoquinovoside. Intensity data were collected at the Australian Synchrotron MX1 beamline the
temperature during data collection was maintained at 100.0(1) using an Oxford Cryosystems cooling
3
8
device. The structure was solved by direct methods and difference Fourier synthesis. Thermal ellipsoid
3
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40
plots were generated using the program ORTEP-3 integrated within the WINGX suite of programs.
Crystal data: C H N O , M = 419.47, T = 130.0 K, l = 0.71073 Å, Orthorhombic, space group P2 2 2 ,
2
4
25
3
4
1 1 1
3
-3
a = 6.9640(14) b = 9.1560(18), c =31.185(6) Å, V = 1988.4(7) Å , Z = 4, D = 1.395 mg M m = 0.215
c
-1
3
mm , q = 32° F(000) = 896, crystal size 0.10 x 0.10 x 0.01 mm , 36298 reflections measured, 6184
max
2
independent reflections [R(int) = 0.098], the final R was 0.0465, [I > 2s(I), 5317 data] and wR(F ) was
0.1276 (all data), GOOF = 1.045, Absolute structure parameter -0.08(4). The structure has been
deposited at the CCDC (deposition number 1884470).
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Acknowledgements
We thank the Australian Research Council (DP180101957) and the National Health and Medical
Research Council (GNT1139549) for financial support , as well as the Australian Synchrotron for
beamtime via the Collaborative Access Program (proposal number 13618b).
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Supporting Information
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Supporting information: Figure S1, X-ray crystallography data and CIF file, H, C{ H} and P{ H}
where relevant) NMR spectra for all new compounds and compounds prepared by new procedures.
(
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